SUMOs (small ubiquitin-related modifiers) are ~100 amino acid proteins that are posttranslationally and covalently conjugated to hundreds of other proteins and thereby regulate a wide range of cellular processes. We have found that sumoylation, like ubiquitination and phosphorylation, is an essential regulator of mitosis in mammalian cells. Mammals express three SUMO paralogs: SUMO-1, SUMO-2 and SUMO-3 (because SUMO-2 and SUMO-3 are 96% identical, they are referred to as SUMO-2/3). Our findings indicate that SUMO-1 and SUMO-2/3 are conjugated to unique subsets of proteins during mitosis and that they regulate distinct processes. However, many unanswered questions still exist. Many of the relevant targets of SUMO-1 and SUMO-2/3 modification remain to be identified and characterized, and mechanisms regulating their temporal modification in mitosis are not known. In addition, the specific molecular effects of SUMO-1 and SUMO-2/3 modification on their targets, and how these effects facilitate progression through mitosis, are still poorly understood. The goals of the research proposed in this renewal grant application are to address these questions and develop a detailed molecular understanding of how sumoylation affects progression through mitosis. These goals will be achieved through four specific aims: (1) We will define the roles that SUMO-2/3 modification and binding play in the targeting and assembly of kinetochore-associated proteins during mitosis. (2) We will identify and characterize molecular mechanisms regulating the temporal and spatial sumoylation of proteins during mitosis. (3) We will identify and characterize chromosome-associated proteins sumoylated during anaphase and telophase. (4) We will investigate the biophysical properties and functions of polymeric SUMO-2/3 chains and mitosis-related chain-binding proteins.